When proteins that contain disulfide bonds are produced in microorganisms they are made in a reduced form that lacks the critical and correct disulfide bonds required for activity. On cell breakage, these proteins are often insoluble because of their non-native conformations and have non-native incorrect disulfide pairing. Various approaches have been used to correctly fold these proteins, which generally require reduction of all disulfides followed by carrying out a controlled oxidation reaction. This controlled oxidation reaction must provide a suitable oxidation environment to oxidatively transform protein thiols into disulfides as well as to allow for the necessary exchange reactions. Conditions that promote effective disulfide interchange require a balance between oxidation and reduction such that the greater thermodynamic stability of the native, or correct, disulfides will be the driving force for achieving the native structure. A consequence of this necessary balance between the need for oxidative drive for disulfide formation and a suitable redox environment for disulfide exchange is that either too high or too low an oxidation environment will compromise the yield of native protein. Examples of incorrectly folded proteins include undesirable disulfide isomers (i.e., with incorrect disulfide pairs) or molecules with intermolecular disulfide pairs forming oligomers of the desired protein products.
Perhaps the most commercially important protein containing disulfide bonds is insulin. U.S. Pat. No. 4,421,685 discloses a process for producing insulin wherein the S-sulfonated form of the A-chain and the S-sulfonated form of the B-chain are reacted with a thiol reducing agent under specific conditions to form insulin. Thiol reducing agents disclosed therein are dithiothreitol (DTT) and dithioerythritol (DTE).